Aqueous solution for use as medium for the specific binding reaction of a binding pair

ABSTRACT

The present invention refers to an aqueous solution for use as medium for the specific binding reaction of a binding pair, wherein a first binding member recognizes its complementary second binding member. The solution contains a) a buffer to control pH; b) a compound A selected from a compound defined by the general formula I R 1 -[[CR 2 R 3 ] p -O] q -R 4 , wherein R 1  is hydrogen or hydroxy group, R 2  for each unit independently is hydrogen or hydroxy group, R 3  is hydrogen, methyl group, or ethyl group, R 4  is hydrogen or alkyl group, p is an integer of from 2 to 10 and q is an integer of from 1 to 100, with the proviso that the compound at least carries two hydroxy groups; a polyol; or saccharide; and c) a non-ionic detergent.

The present invention relates to an aqueous solution for the use asmedium for the specific binding reaction of a binding pair.

BACKGROUND OF THE INVENTION

Immunoassays where one or more antibodies are used to detect the testsubstance (analyte) in a test sample are widely known. The evolution ofimmunoassay methods increased the sensitivity of this test. Despite ofthe developments within the recent decades, there remains a desire toeliminate unspecific binding reactions, cross-reactivities and theinfluence of the compounds present in the matrix.

Immunoassays depend upon the ability of a first binding member of abinding member pair, e.g. an antigen or a ligand, to specifically bindto a second binding member of a binding member pair, e.g. an antibody ora receptor. In order to determine the extend of such binding, aconjugate, comprising one of such binding members are labeled with adetectable moiety. Such binding member pairs can be an antigen and anantibody directed to such an antigen.

Immunoassays can be performed in a competitive immunoassay format or ina sandwich immunoassay format. In the competitive immunoassay format anantigen can be immobilized to a solid phase material whereby the amountof detectable moiety that is bound to a solid phase material can bedetected, measured and correlated to the amount of antibody present inthe test sample. Examples of solid phase materials include beads,particles, micro-particles and the like. In the sandwich immunoassayformat a test sample, containing for example an antibody, is contactedwith a protein such as an antigen. The antigen is immobilized on a solidphase material. Examples of solid phase materials include beads,particles, micro-particles and the like. The solid phase material istypically created with a second antigen or antibody that has beenlabeled with a detectable moiety. The second antigen or antibody,respectively, then becomes bound to the corresponding antibody orantigen, respectively, on the solid phase material and, after one ormore washing steps, to remove any unbound material an indicator materialsuch as a chromogenic substance, is introduced to react with thedetectable moiety to produce the detectable signal. e.g. a color change.The color change is then detected, measured and correlated to the amountof antibody present in the test sample. It should also be noted thatvarious dilutents and buffers are also required to optimize theoperation of the micro-particles, antigens, conjugates and othercomponents of the assay that participate in chemical reactions.

In order to achieve optimal results in immunoassays the solution whichis used for the binding reactions between the binding partners (forexample the antibody and antigen reaction or the complex formation ofligand and receptor) must provide a medium that optimizes the ability ofantibodies to bind to the antigen, or must provide a medium thatoptimizes the ability of ligands to bind to the receptor, whilenon-specific interactions, low-affinity binding and matrix effects arestrongly reduced or even prevented in order to avoid the generation of afalse signal.

In order to eliminate non-specific interactions and cross-reactivitiesdetergents have been added to buffers which are used for washing stepsafter the binding reaction in order to remove unspecific bindings.

For immunoassays, like western-blot analyses, enzyme-linkedimmuno-sorbant assay (ELISA) and others, solutions containing phosphatebuffered saline (PBS) supplemented with bovine serum albumin and 0.01 to0.05 (v/v) Tween® 20 is used as medium for the binding reactions betweenthe binding partners (for example antibody and antigen). It is, however,very often experienced that unspecific or low-affinity binding,cross-reactivities and matrix effects can not be avoided with suchbuffers of the state of art. For example, when developing a CRP-assayinvolving the detection of a plurality of analytes it appeared thatcross-reactivities due to the use of the plurality of antibodies as wellas matrix effects became a problem, which could not be solved by the useof conventional immunoassay buffers.

The object of the present invention therefore was to provide a solutionfor the use as a medium for the specific binding reaction of a bindingmember pair wherein the unspecific binding, low affinity binding,cross-reactivities and matrix effects are strongly reduced or evenprevented. Furthermore, it was the object of the present invention toprovide a method of an immunoassay, wherein unspecific and low affinitybinding, cross-reactivities and matrix effects are reduced or prevented.

SUMMARY OF THE INVENTION

The object of the present invention is solved by an aqueous solution foruse as medium for the specific binding reaction of a binding memberpair, wherein a first binding member recognizes its complementary secondbinding member, the solution comprising

a) a buffer to control pH;

b) a compound A selected from the group consisting of:

-   -   a compound defined by the general formula I        R¹-[[CR²R³]_(p)-O]_(q)-R⁴, wherein R¹ is hydrogen or hydroxy        group, R² for each unit Independently is hydrogen or hydroxy        group, R³ is hydrogen, methyl group, ethyl group, R⁴ is hydrogen        or alkyl group, p is an integer of from 2 to 10 and q is an        integer of from 1 to 100, with the proviso that the compound at        least carries two hydroxy groups;    -   polyol;    -   saccharide;

c) a non-ionic detergent.

In case R⁴ in the general formula I of compound A is hydrogen theneighbouring residue R² is also hydrogen. In case R¹ is hydroxy groupthe neighbouring residue R² is hydrogen. In a preferred embodiment q inthe formula of compound A is an Integer of from 1 to 50, more preferredfrom 1 to 30.

The inventors of the present invention surprisingly have found that theaqueous solution according to the present invention reduces thecross-reactivities, matrix effects, unspecific bindings and lowaffinity-binding in immunoassays. Furthermore, it was found that eveneffects due to heterophilic antibodies (human anti-mouse-antibody) areprevented when the aqueous solution according to the present inventionis used. Additionally, negative effects due to rheuma factors,hemoglobin, bilirubin and triglycerides can be avoided with this buffereven in case of plasma applications.

As used herein a “binding member pair” comprises a “first binding memberand a “second binding member”. Both binding members undergo a specificbinding to each other. The first binding member of a binding member pairmay be an antigen or a ligand, respectively. The second binding member(e.g. an antibody or a receptor, respectively) specifically recognizesand binds to the first binding member (e.g. antigen or ligand,respectively). The second binding member is the corresponding bindingmember and therefore also named “corresponding binding member”. Theartisan will understand that the terms “first” binding member and“second” binding member, respectively, may be for example the antigenand the corresponding antibody, respectively, or vice versa.

The aqueous solution according to the present invention represents auniversal buffer as a medium in immunoassays and binding reactions in avariety of matrices, for example blood plasma, blood serum and others.In case of multi-analyte applications, for example, if protein chips areused, the simultaneous incubation of several (or a plurality) ofanalytes and several antibodies, unspecific bindings andcross-reactivities very often occur. Such undesired binding reactionshave been observed in many cases. The use of standard ELISA buffersknown in the state of art could not prevent such cross-reactivityeffects. In addition in the state of art, the use of native samplesresulted in matrix effects which gave erroneous measurements compared toother methods for reference. As used herein the term “matrix” refers toall compounds present in a native sample, like blood serum; inparticular the term “matrix” refers to the organic and especially tobiological compounds such as proteins.

The aqueous solution according to the present invention may be used asmedium for the binding reaction of a binding pair, as sample dilutionbuffers for immunoassays and binding reactions as well as dilutionbuffer for antibodies and antigens, respectively. Further applicationsare multi-analyte immunoassays and proteomics, wherein undesiredcross-reactivities of antibodies labeled with the fluorophor can beprevented. Fluorophor-labeled antibodies tend to bind other proteins inan unspecific manner. By using the aqueous solution according to thepresent invention such effects can be avoided.

By using the buffer according to the present invention in immunoassayslike ELISA and protein chips a further positive effect was shown. Whenincubating the surface carrying the immobilized antibodies with thebuffer according to the present Invention the activity of theimmobilized antibodies was increased. This is resulting in anenhancement of the binding of the analyte to the immobilized antibody.In conclusion, the buffer according to the present invention in additionto the reduction of unspecific signals and unspecific effects alsoincreases the specific signals due to a positive influence on thespecific binding reaction between the analyte and the antibody. Theresulting increase of the activity of the immobilized antibodiesprovides a higher sensitivity of the respective assay.

The buffer according to the present invention may be used forimmunoassays ELISA, EIA, FIA, lateral-flow-test, protein chips,multi-analyte assays, western blots, dot blots, immunohistochemistry,receptor-ligand-assays and immuno-PCR.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment of the present Invention the aqueous solutionis further comprising a protein In an amount effective toimmunologically block non-specific antibody binding. This proteinpreferably is selected from the group bovine serum albumin, ovalbumin,casein, fetal bovine serum. Further preferred, the protein is present inthe aqueous solution in a concentration in the range of 0.1 to 2% (w/v)and further preferred in the range of 0.5 to 1.5% (w/v). These proteinsare not recognized by any of the antibodies used in the immunoassays.This unrecognized protein allows the immunological blocking ofnon-specific antibody-binding by molecules or compounds which might bepresent in the sample.

In a further embodiment the aqueous solution Is comprising a saltselected from the group NaCl, KCl, NH₄Cl. Further preferred the aqueoussolution is having an ionic strength of 100 mM to 1.5 mM, more preferredof 200 mM to 1 M, even further preferred of 200 mM to 800 mM,particularly more preferred of 200 mM to 600 mM and most preferred of250 mM to 500 mM. The inventors surprisingly have found that a highionic strength of the buffer used as a medium for binding reactions, forexample, in the range of 200 mM to 600 mM is further reducing unspecificbinding and cross-reactivies, while the speck binding reaction is notnegatively influenced.

In a particular preferred embodiment the buffer of the aqueous solutionis selected from the group Tris (Tris(hydroxymethyl)-aminomethane, Pipes(Piperazine-1,4-bis-2-ethane sulfonic acid), Mes (4-Morpholino ethanesulfonic acid), Hepes (4-(2-hydroxyethyl)-1-piperazine-ethane sulfonicacid), phosphate buffer.

In a further preferred embodiment the compound A is selected from thegroup polyalkylene glycol, polypropylene glycol, propylene glycol,polyethylene glycol, ethylene glycol, monosaccharides, disaccharides,trisaccharides, saccharose, mannose, trehalose, polyol, glycerol andmixtures thereof. In a preferred embodiment the concentration of thecompound A is in the range of 0.5 to 25% (v/v), preferably in the rangeof 2.0 to 20% (v/v), more preferred in the range of 2.0 to 15% (v/v),further more preferred in the range of 2.0 to 10% (v/v), even morepreferred in the range of 2.0 to 7% (v/v), and most preferred around 5%(v/v). The concentration is given in % (v/v) in case coumpound A is aliquid. In case the compound A is solid (for example a saccharide) theconcentration has to be understood as % (w/v).

In a further preferred embodiment the aqueous solution comprises asnon-ionic detergent a compound of the general formula selected from thegroup

a) a substituted phenyl residue having substituents R¹ and R²(R¹-Ph-R²), wherein R¹ is C₁-C₉ alkyl group, and R² is—O—[CH₂-CH₂-O]_(a)-H group, wherein “a” is an integer of 5 to 40,wherein R² in respect to R¹ is in para, meta or ortho position.b)

wherein n, x, y and z together is an integer of 5 to 40, R is a fattyacid residue.

Further preferred the non-ionic detergent is selected from the groupDodecylpoly(ethyleneglycolether)_(m), wherein m is an integer of 5 to40; 1-O-n-Octyl-β-D-glucopyranoside (n-Octylglucoside);Alkylphenolpoly(ethyleneglycol-ether)_(m), wherein m is an integer of 5to 40, preferably m=11 (Nonidet P40®); 1-O-n-Dodecyl-β-D-glucopyranosyl(1-4)alpha-D-glucopyranoside; Dodecylpoly-(ethyleneglycolether)_(m),wherein m is an integer of 5 to 40, preferably m=23 (Brij35®);Poly(oxyethylene)(20)-sorbitane mono fatty acid ester, preferablyselected from Poly(oxyethylene)(20)-sorbitane monooleate (Tween®80),Poly(oxyethylene)(20)-sorbitane monolaurate (Tween®20),Poly(oxyethylene)(20)-sorbitane monopalmitat (Tween®40),Poly(oxyethylene)(20)-sorbitane monostearate);Octylphenolpoly(ethyleneglycoiether)_(m), wherein m is an integer of 5to 40, preferably m=10 (Triton®X-100).

In preferred embodiments the concentration of the non-ionic detergent isin the range of 0.1 to 1.0% (v/v). Preferably, the concentration of thenon-ionic detergent in the range of 0.15 to 1.0% (v/v), more preferredin the range of 0.2 to 1.0% (v/v), further more preferrred in the rangebetween 0.2 and 0.8% (v/v), even more preferred in the range of 0.25% to0.6% (v/v), and most preferred about 0.25% (v/v).

An important feature of the present invention is the presence ofcompound A as given in claim 1 in combination with the non-ionicdetergent. Each of said both ingredients (compound A and non-ionicdetergent) is present in the aqueous solution of the present inventionin higher concentrations as it is known in respect to incubationsolutions for immunoassays of the state of art. In a further preferredembodiment an aqueous solution is provided, wherein the ratio of thenon-ionic detergent to the compound A is from 1:15 to 1:25, preferablyaround 1:20.

In a particularly preferred embodiment the aqueous solution comprisescompound A in a concentration in the range of 2 to 7% (v/v) and anon-ionic detergent in the range of between 0.2 to 0.8% (v/v).Preferably the aqueous solution is having an ionic strength of 200 mM to1 M, more preferred of 200 mM to 800 mM, particularly more preferred of200 mM to 600 mM and most preferred of 250 mM to 500 mM. It is preferredthat the aqueous solution comprises as compound A a compound selectedfrom the group polyalkylene glycol, polypropylene glycol, propyleneglycol, polyethylene glycol, ethylene glycol, glycerol and mixturesthereof, more preferred a compound selected from polypropylene glycol,propylene glycol, polyethylene glycol, ethylene glycol and mostpreferred ethylene glycol.

The aqueous solution according to the present invention preferably doesnot contain dithiothreitol. Further preferred the aqueous solutionaccording to the present invention does not contain β-mercapto-ethanol.

In a further preferred embodiment the pH of the aqueous solution isadjusted in the range of 5.6 to 9.6, preferably in the range of 6.0 to9.0, further preferred in the range of 6.5 to 8.0 and most preferred inthe range of 6.8 to 7.4.

A particularly preferred embodiment of the aqueous solution is havingthe capability of reducing unspecific binding, cross-reactivity anddisturbing effects of the matrix. The aqueous solution according to thepresent invention particularly has the capability of preventing thelow-affinity binding with K_(D) values of up to 10⁻⁷ M, compared tostandard conditions. Further preferred the aqueous solution has thecapability of preventing the low-affinity binding with K_(D) values ofup to 10⁻⁷ M and reducing the mid-range affinity binding with K_(D)values in the range of between 10⁻⁷ M and 10⁻⁸ M by at least 90%compared to standard conditions. Even further preferred, the aqueoussolution has the capability of preventing the low-affinity binding withK_(D) values of up to 10⁻⁷ and reducing the mid-range affinity bindingwith K_(D) values in the range of between 10⁻⁷ and 10⁻⁹ by at least 90%compared to standard conditions. As used herein “standard conditions”are represented by an aqueous solution consisting of 50 mM PBS(phosphate buffered saline, pH 7.4), 150 mM NaCl, 1% (w/v) BSA (see alsotable 1: reference example). The same results were obtained whencomparing the measurements with another standard solution, namely anaqueous solution consisting of 50 mM PBS (pH 7.4), 100 mM NaCl, 0.05%(v/v) Tween®20.

Apart from the effect of reducing the unspecific, low affinity bindingas well as matrix effects, the aqueous solution particularly preferredis having the capability to increase the binding activity of antibodies,preferably the binding activity of immobilized antibodies as well as thebinding activity between ligands and receptors. The increase of thebinding activity of immobilized antibodies by using the solutionaccording to the present Invention was about 10% or more.

The object of the present invention is also solved by a concentrate ofthe aqueous solution of the present invention described before,preferably a 2 to 10 fold concentrate, more preferred a 3 to 5 foldconcentrate.

Further, the object of the present invention is solved by the use of theaqueous solution according to the present invention as a medium for thebinding reaction of a binding pair, wherein a first binding memberspecifically recognizes and binds its complementary second bindingmember. Preferably the aqueous solution is used as a medium for theantibody-antigen binding reaction and in an alternative embodiment theaqueous solution is used as a medium for the receptor-ligand bindingreaction. In other preferred embodiments the aqueous solution is used asdilution buffer for samples, reagents, ligands, receptors, antigens,antibodies. The aqueous solution may also preferably be used as awashing buffer in immunoassays after the binding reaction was carriedout.

The invention further provides a method for reducing unspecific bindingand/or cross-reactivity and/or disturbing effects of matrices during aspecific binding reaction of a binding pair, wherein a first bindingmember recognises its complementary second binding member, the methodcomprising the use of the aqueous solution of the present invention asmedium for the specific binding reaction.

In another aspect of the invention the aqueous solution according to thepresent invention can be provided as a component of a kit. As usedherein, the term “kit” means a collection of reagents and associatedmaterials e.g. buffers, carrier comprising an immobilised binding memberand reagents which are required to perform an assay. Therefore, thepresent invention provides a kit for detection by immunoassay of atleast one analyte to be tested, wherein the analyte to be tested is afirst binding member of a binding member pair, wherein the first bindingmember binds specifically to its complementary binding member, the kitcomprising:

-   -   a) a vessel containing an aqueous solution of the present        invention;    -   b) a carrier comprising the complementary binding member        immobilised thereon to capture the analyte; and    -   c) optionally, a reagent which immunologically recognises the        analyte bound to the complementary binding member, wherein the        reagent (antibody) is conjugated to a means of detection; and    -   d) optionally: reagents which are reactive with said means of        detection to produce a detectable reaction product.

A typical kit for example is used as ELISA kit for the detection of, forexample, antibodies in blood serum. In this case the carrier accordingto b) comprises as complementary binding member immobilised thereon, forexample, a virus antigen to capture the analyte. The analyte which isthe antibody in the blood serum. The reagent according to c) whichImmunologically recognises the analyte then is an anti-antibody which isrecognising the captured antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is characterized in that the aqueous solution foruse as medium for the specific binding reaction of a binding memberpair, wherein a first binding member recognizes its complementary secondbinding member, comprises:

a) a buffer to control pH;

b) a compound A selected from the group consisting of:

-   -   a compound defined by the general formula I        R¹-[[CR²R³]_(p)-O]_(q)-R⁴, wherein R¹ is hydrogen or hydroxy        group, R² for each unit independently Is hydrogen or hydroxy        group, R³ is hydrogen, methyl group, ethyl group, R⁴ is hydrogen        or alkyl group, p is an integer of from 2 to 10 and q Is an        integer of from 1 to 100, with the proviso that the compound at        least carries two hydroxy groups;    -   polyol;    -   saccharide;

c) a non-ionic detergent.

The method of the present invention for reducing unspecific bindingand/or cross-reactivity and/or disturbing effects of matrices during aspecific binding reaction of a binding pair, wherein a first bindingmember recognises its complementary second binding member, i.e. for aimmunoassay, is characterized to comprise the use of the above aqueoussolution.

Preferably an aqueous solution was found to be useful which comprisescompound A in a concentration in the range of 2 to 7% (v/v) and anon-ionic detergent in the range of between 0.2 to 0.8% (v/v).Preferably the aqueous solution is having an ionic strength of 200 mM to1 M, more preferred of 200 mM to 800 mM, particularly more preferred of200 mM to 600 mM and most preferred of 250 mM to 500 mM. It is preferredthat the aqueous solution comprises as compound A a compound selectedfrom the group polyalkylene glycol, polypropylene glycol, propyleneglycol, polyethylene glycol, ethylene glycol, glycerol and mixturesthereof, more preferred a compound selected from polypropylene glycol,propylene glycol, polyethylene glycol, ethylene glycol and mostpreferred ethylene glycol.

The present inventions will be explained in more detail in the followingexamples. However, the examples are only used for illustration and donot limit the scope of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the amount of detector antibody C6 bound to CRP in asandwich assay as absorbance at 450 nm plotted against the concentrationof CRP [ng/ml]. The binding reaction of detector antibody C6 to theprotein CRP was performed as outlined under Example 1 under standardconditions and by using sample buffers according to the presentinvention, respectively (see table 1). Sample buffers Ito III accordingto the solution of the present invention reduce the influence of thematrix effects very well. Sample buffer I shows the best sensitivity.The low sensitivity of the reference buffer is caused by a strong matrixeffect.

FIG. 2 shows the influence of two different buffers on high backgroundsignals caused by unspecific binding of the polyclonal detector antibodyP2 which binds unspecific to the capture antibody P3 (according toExample 2). In this experiment no analyte was present. Sample buffer Idecreases significantly the unspecific binding compared to the highbackground signals with reference example buffer II.

EXAMPLES Example 1 Reduction of Matrix Effects

100 μl diluted capture antibody C2 (final concentration 1 μg/ml inPBS-Buffer) was added to each well of a microtiter plate (C8 StarWellModule, NUNC) and the plate was covered with a plate sealer. The captureantibody is directed against CRP (c-reactive protein). Then the platewas incubated for 5 hours at room temperature. The plate sealer wasremoved and the plate was washed 4 times with 300 μl washing buffer (10mM Phosphate, 350 mM NaCl, 0.05% Tween, pH 7.4) per well. Then 200 μl ofblocking solution (PBS-Buffer pH 7.4, 1% BSA) was added to each well.After covering with a plate sealer the plate was incubated overnight at4° C. The analyte CRP (c-reactive protein) was diluted In rabbit serum(0-5 ng/ml) and Incubated for 30 min at room temperature. Thebiotin-labeled detector antibody C6 (directed against CRP) was dilutedin different sample buffers and reference example buffers (see table 1).The final concentration was 4 μg/ml in each preparation. TheCRP-containing rabbit serum standards were diluted 1:2 with detectorantibody containing sample buffers. The preparations were incubated for30 min at room temperature. The plate sealer was removed and the platewas washed 4 times with 300 μl washing buffer. Oddments of washingbuffer were completely removed by taping the plate dry. 100 μl of theCRP-preparations were added to the wells. The plate was covered with aplate sealer and incubated for 4 h at room temperature under gentleshaking. After that the plate was washed again. 100 μl of dilutedNeutrAvidin™-Horseradish peroxidase conjugated (final concentration 0.05μg/ml in PBS-Buffer) was added to each well. The plate was incubated for1 h at room temperature under gentle shaking. Then the plate was washedagain. Equal volumes of the two solutions of ImmunoPure®TMB Substratwere mixed and 100 μl were added immediately to each well. The plate wasincubated at room temperature until the desired colour developed. Thecolour changed from clear to brilliant blue. In a final step thereaction was stopped by adding 150 μl 2 M H₂SO₄ to each well and theabsorbance was read out at 450 nm with an ELISA plate reader (Moleculardevices). The influence of different buffers on matrix effects areplotted in FIG. 1. Table 1 shows the results of the test. In table 1 thereduction of non-specific binding, low-affinity binding and matrixeffects is indicated in the column “result” with “+”. The number of “+”is indicating the amount of reduction of non-specific binding,low-affinity binding and matrix effects compared to the referenceexample buffer (“−”). FIG. 1 shows the amount of detector antibody C6bound to CRP as absorbance at 450 nm plotted against the concentrationof CRP [ng/ml]. Sample buffer I shows the best sensitivity. The lowsensitivity of the reference buffer is caused by a strong matrix effect.

Example 2 Reduction of an Unspecific Binding of a Polyclonal DetectorAntibody

For the next assay 250 μl diluted capture antibody P3 (polyclonalrabbit-anti-protease, own preparation, final concentration 1 g/ml inPBS-Buffer) was added to each well of a microliter plate (C8 StarWellModule, NUNC) and the plate was covered with a plate sealer. The platewas incubated for 4 hours at room temperature. After that the platesealer was removed and the plate was washed 4 times with 300 μl washingbuffer (10 mM Phosphate, 350 mM NaCl, 0.05% Tween®, pH 7.4) per well.Then 200 μl of blocking solution (PBS-Buffer pH 7.4, 1% BSA) was addedto each well. The plate was covered again with a plate sealer andincubated overnight at 4° C. The biotin-labeled polyclonal detectorantibody P2 (polyclonal rabbit-anti-protease, own preparation) wasdiluted in reference example buffer II or in sample buffer I (seetable 1) respectively. (final concentration 10 μg/ml in eachpreparation) and added to the wells (250 μl per well; fivefoldreplicates). The plate was incubated for 2 h at room temperature. Theplate sealer was removed and the plate was washed 4 times with 300 plwashing buffer. Oddments of washing buffer were completely removed bytaping the plate dry. 250 μl of diluted NeutrAvidin™-Horseradishperoxidase conjugated (final concentration 0.5 μg/ml in PBS-Buffer) wasadded to each well. The plate was incubated for 1 h at room temperatureunder gentle shaking. Then the plate was washed again. Equal volumes ofthe two solutions of ImmunoPure®TMS Substrat were mixed and 100 μl wereadded immediately to each well. The plate was Incubated at roomtemperature until the desired colour develops. The colour changes fromclear to brilliant blue. In a final step the reaction was stopped byadding 150 μl 2 M H₂SO₄ to each well and the absorbance was read out at450 nm with an ELISA plate reader (Molecular devices). The influence ofthe two different buffers on high background signals caused byunspecific binding of the polyclonal detector antibody P2 which bindsunspecifically to the capture antibody P3 is plotted in FIG. 2. FIG. 2shows the reduction of background signals due to the use of samplebuffer I according to the present invention. In this experiment noanalyte was used. Further, as antibody a polyclonal serum was used. Apolyclonal serum comprises many different antibodies directed against atarget protein. Many antibodies will bind with low or lower affinity,while some antibodies will bind with mid-range affinity and one or onlya few antibodies will bind with high affinity. The use of the bufferaccording to the Invention prevents the low-affinity binding and atleast reduces the mid-range affinity binding of the respectiveantibodies as shown in FIG. 2. As a result, once an analyte will beadded in such an assay the signal-to noise-ratio will be improved due tothe properties of the aqueous solution according to the presentinvention.

TABLE 1 The table shows the results of the test according to Example 1:Result (=Reduction of non-specific, low-affinity binding and matrixeffects) Compound A Non-ionic detergent solution Buffer/pH[concentration] [concentration] NaCl BSA Result reference example PBS pH7.4 — — 150 mM 1% − (state of art) sample buffer I Tris pH 7.4   5%ethylene glycol 0.25 Tween ®20 300 mM 1% ++++ sample buffer II Tris pH7.4 0.5% ethylene glycol  0.1 Tween ®20 150 mM 1% + sample buffer IIIPBS pH 7.4   3% glycerol 0.15 Triton X100 200 mM 1% ++ sample buffer IVTris pH 7.4   5% glycerol 0.25 Triton X100 300 mM 1% ++++ sample bufferV PBS pH 7.4   3% glycerol 0.15 Triton X100 600 mM 1% +++ referenceexample PBS pH 7.4 — 0.05 Tween ®20 150 mM 1% −/+ II (state of art)sample buffer VI Tris pH 7.4   5% ethylene glycol 0.25 Tween ®20 — — +++sample buffer VII Tris pH 7.4   5% polyethylene glycol 0.25 Tween ®20 —— +++ sample buffer VIII Tris pH 7.4   5% polypropylene glycol 0.25Tween ®20 — — +++ sample buffer IX Tris pH 7.4   5% polyethylene glycol0.25 Tween ®20 300 mM — ++++ sample buffer X Tris pH 7.4   5%polypropylene glycol 0.25 Tween ®20 300 mM — ++++ sample buffer XI TrispH 7.4   5% polyethylene glycol 0.25 Tween ®20 300 mM 1% ++++ samplebuffer XII Tris pH 7.4   5% polypropylene glycol 0.25 Tween ®20 300 mM1% ++++ sample buffer XIII Tris pH 7.4   5% propylene glycol 0.25Tween ®20 — — +++ sample buffer XIV Tris pH 7.4   5% propylene glycol0.25 Tween ®20 300 mM — ++++ sample buffer XV Tris pH 7.4   5% propyleneglycol 0.25 Tween ®20 300 mM 1% ++++ sample buffer XVI PBS pH 7.4   5%ethylene glycol 0.25 Triton X100 — — +++ sample buffer XVII PBS pH 7.4  5% ethylene glycol 0.25 Triton X100 300 mM — ++++ sample buffer XVIIIPBS pH 7.4   5% ethylene glycol 0.25 Triton X100 300 mM 1% ++++ samplebuffer XIX PBS pH 7.4   5% glycerol 0.25 Triton X100 — — + sample bufferXX PBS pH 7.4   5% glycerol 0.25 Triton X100 300 mM — +++ sample bufferXXI PBS pH 7.4   5% glycerol 0.25 Triton X100 300 mM 1% +++ samplebuffer XXII PBS pH 7.4   5% trehalose 0.25 Triton X100 — — + samplebuffer XXIII PBS pH 7.4   5% trehalose 0.25 Triton X100 300 mM — ++sample buffer XXIV PBS pH 7.4   5% trehalose 0.25 Triton X100 300 mM 1%++

What is claimed is :
 1. A method for identifying the presence of anantigen or an antibody in a sample, the method comprising: contactingthe sample with a complementary capture antibody or a capture antigen inan aqueous solution, specifically binding the antigen or the antibody tothe complementary capture antibody or capture antigen, respectively; anddetecting the presence of the antigen or antibody bound to thecomplementary capture antibody or capture antigen with a detectorantibody that binds to either (a) the antigen, which is bound to thecomplementary capture antibody, or (b) the antibody, which is bound tothe capture antigen, said sample further comprising a sample componentcomprising an interfering protein other than an antibody or atriglyceride, wherein said sample component interferes with the specificbinding between the antigen or the antibody in the sample and thecomplementary capture antibody or capture antigen or detector antibody;said aqueous solution comprising: a) a buffer to control pH; b) acompound A selected from the group consisting of: a compound defined bythe general formula I R¹-[[CR²R³]_(p)-O]_(q)-R⁴, wherein R¹ is hydrogenor hydroxy group, R² for each unit independently is hydrogen or hydroxygroup, R³ is hydrogen, methyl group, ethyl group, R⁴ is hydrogen oralkyl group, p is an integer of from 2 to 10 and q is an integer of from1 to 100, with the proviso that the compound at least carries twohydroxy groups; a polyol; and a saccharide; and c) a non-ionicdetergent; and wherein said aqueous solution reduces the binding of saidinterfering sample component in the sample to any one or more of thefollowing: complementary capture antibody or capture antigen or detectorantibody or the antigen or the antibody of the sample to be detected,thereby reducing interference by the interfering sample component withthe specific binding of said antigen or antibody to the complementarycapture antibody or capture antigen or detector antibody, compared toconducting said specific binding in the absence of said aqueoussolution.
 2. The method of claim 1, wherein the aqueous solution has anionic strength of 100 mM to 800 mM.
 3. The method of claim 1, whereinthe aqueous solution has an ionic strength of 100 mM to 600 mM.
 4. Themethod of claim 1, wherein the aqueous solution has an ionic strength of100 mM to 500 mM.
 5. The method of claim 1, wherein the aqueous solutionhas an ionic strength of 200 mM to 600 mM.
 6. The method of claim 1,wherein the aqueous solution has an ionic strength of 250 mM to 600 mM.7. The method of claim 1, wherein the solution comprises a salt selectedfrom the group consisting of NaCl, KCl, and NH₄Cl.
 8. The method ofclaim 1, wherein the concentration of compound A is from 2.0-25%,wherein the concentration of compound A is in terms of % (v/v) whencompound A is a liquid or the concentration of compound A is in terms of% (w/v) when compound A is a solid.
 9. The method of claim 1, whereinthe concentration of the non-ionic detergent is from 0.1-1.0%.
 10. Themethod of claim 1, wherein said aqueous solution further comprises ablocking protein in an amount effective to immunologically blocknon-specific antibody binding.
 11. The method of claim 10, wherein theblocking protein is selected from the group consisting of bovine serumalbumin, ovalbumin, casein, and fetal bovine serum.
 12. The method ofclaim 10, wherein the concentration of the blocking protein is in therange of 0.1 to 2% w/v.
 13. The method of claim 1, wherein the buffer isselected from the group consisting of Tris(Tris(hydroxymethyl)-aminomethane, Pipes (Piperazine-1,4-bis-2-ethanesulfonic acid), Mes (4- Morpholino ethane sulfonic acid), Hepes(4-(2-hydroxyethyl)-1-piperazine- ethane sulfonic acid), and phosphatebuffer.
 14. The method of claim 1, wherein compound A is selected fromthe group consisting of polyalkylene glycol, polypropylene glycol,propylene glycol, polyethylene glycol, ethylene glycol, monosaccharides,disaccharides, trisaccharides, saccharose, mannose, trehalose, polyol,glycerol and mixtures thereof
 15. The method of claim 1, wherein thenon-ionic detergent is a compound selected from the group consisting of:a) a substituted phenyl residue having substituents R¹ and R²(R¹-Ph-R²), wherein R¹ is C₁-C₉ alkyl group, and R² is a—O—[CH₂-CH₂-O]_(a)-H group, wherein “a” is an integer of 5 to 40,wherein R² in respect to R¹ is in para, meta or ortho position, b)

wherein n, x, y and z together is an integer of 5 to 40, R is a fattyacid residue; c) Dodecylpoly(ethyleneglycolether)_(m), wherein m is aninteger of 5 to 40; d) 1-O-n-Octyl-β-D-glucopyranoside(n-Octylglucoside); and e) 1-O-n-Dodecyl-β-D-glucopyranosyl(1-4)alpha-D-glucopyranoside.
 16. The method of claim 1, wherein thenon-ionic detergent is selected from the group consisting ofAlkylphenolpoly(ethylene-glycolether)_(m), wherein m is an integer of 5to 40; Dodecylpoly-(ethyleneglycolether) _(m), wherein m=23 (Brij35®);Poly(oxyethylene)(20)-sorbitane mono fatty acid ester;Poly(oxyethylene)(20)-sorbitane monooleate (Tween®80); Poly(oxyethylene)(20)-sorbitane monolaurate (Tween®20); Poly(oxyethylene)(20)-sorbitanemonopalmitate (Tween®40); Poly(oxyethylene)(20)-sorbitane monostearate);Octylphenolpoly(ethylene-glycolether)_(m), wherein m is an integer of 5to
 40. 17. The method of claim 1, wherein the non-ionic detergent isselected from the group consisting ofAlkylphenolpoly(ethylene-glycolether)_(m), wherein m=11 (Nonidet P40®);and Octylphenolpoly(ethylene-glycolether)_(m), wherein m=10 (Triton®X100).
 18. The method of claim 1, wherein the ratio of the non-ionicdetergent to the compound A is from 1:15 to 1:25;
 19. The method ofclaim 1, wherein the aqueous solution does not contain dithiothreitol.20. The method of claim 1, wherein the pH is adjusted in the range of5.6 to 9.6.
 21. The method of claim 1, wherein the aqueous solution hasthe capability of preventing binding of heterophilic antibodies withK_(D) values of up to 10⁻⁷ M.
 22. The method of claim 1, wherein theaqueous solution has the capability of preventing binding ofheterophilic antibodies with K_(D) values of up to 10⁻⁷ M and reducingthe mid-range affinity binding with K_(D) values in the range of between10⁻⁷ M and 10⁻⁸ M by at least 90%.
 23. The method of claim 1, whereinthe aqueous solution has the capability of preventing binding ofheterophilic antibodies with K_(D) values of up to 10⁻⁷ M and reducingthe mid-range affinity binding with K_(D) values in the range of between10⁻⁷ and 10⁻⁹ by at least 90%.
 24. The method of claim 1, wherein thesample is blood plasma or blood serum.
 25. The method of claim 1,wherein the antigen to be identified is C-reactive protein.
 26. Themethod of claim 1, wherein the interfering protein is selected from thegroup consisting of rheuma factors, hemoglobin and bilirubin.